26,27-homologated-20-EPI-2-alkylidene-19-nor-vitamin D compounds

ABSTRACT

This invention provides a novel class of vitamin D related compounds, namely, the 2-alkylidene-19-nor-vitamin D derivatives, as well as a general method for their chemical synthesis. The compounds have the formula: 
     
       
         
         
             
             
         
       
     
     where Y 1  and Y 2 , which may be the same or different, are each selected from the group consisting of hydrogen and a hydroxy-protecting group, R 6  and R 8 , which may be the same or different, are each selected from hydrogen, alkyl, hydroxyalkyl and fluoroalkyl, or when taken together represent the group —(CH 2 ) X — where X is an integer from 2 to 5, and where the group R represents any of the typical side chains known for vitamin D type compounds. These 2-substituted compounds are characterized by relatively high intestinal calcium transport activity and relatively high bone calcium mobilization activity resulting in novel therapeutic agents for the treatment of diseases where bone formation is desired, particularly low bone turnover osteoporosis. These compounds also exhibit pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as anti-cancer agents and for the treatment of diseases such as psoriasis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.09/370,966 filed Aug. 10, 1999, which in turn is a continuation of Ser.No. 09/151,113, filed Sep. 10, 1998, now U.S. Pat. No. 5,936,133, whichin turn is a division of application Ser. No. 08/819,693, filed Mar. 17,1997, now U.S. Pat. No. 5,843,928.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agencies:

-   -   NIH DK 14881-26S1        The United States has certain rights in this invention.

BACKGROUND OF THE INVENTION

This patent invention relates to vitamin D compounds, and moreparticularly to vitamin D derivatives substituted at the carbon 2position.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, and morerecently their activity in cellular differentiation has beenestablished, Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987).Many structural analogs of these metabolites have been prepared andtested, including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, variousside chain homologated vitamins and fluorinated analogs. Some of thesecompounds exhibit an interesting separation of activities in celldifferentiation and calcium regulation. This difference in activity maybe useful in the treatment of a variety of diseases such as renalosteodystrophy, vitamin D-resistant rickets, osteoporosis, psoriasis,and certain malignancies.

Recently, a new class of vitamin D analogs has been discovered, i.e. theso called 19-nor-vitamin D compounds, which are characterized by thereplacement of the A-ring exocyclic methylene group (carbon 19), typicalof the vitamin D system, by two hydrogen atoms. Biological testing ofsuch 19-nor-analogs (e.g., 1α,25-dihydroxy-19-nor-vitamin D₃) revealed aselective activity profile with high potency in inducing cellulardifferentiation, and very low calcium mobilizing activity. Thus, thesecompounds are potentially useful as therapeutic agents for the treatmentof malignancies, or the treatment of various skin disorders. Twodifferent methods of synthesis of such 19-nor-vitamin D analogs havebeen described (Perlman et al., Tetrahedron Lett. 31, 1823 (1990);Perlman et al., Tetrahedron Lett. 32, 7663 (1991), and DeLuca et al.,U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

Recently, 2-substituted analogs of 1α,25-dihydroxy-19-norvitamin D₃ havealso been synthesized, i.e. compounds substituted at 2-position withhydroxy or alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), whichexhibit interesting and selective activity profiles. All these studiesindicate that binding sites in vitamin D receptors can accommodatedifferent substituents at C-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, their analogs which are characterized bythe presence of an alkylidene (particularly methylene) substituent atthe carbon 2 (C-2), i.e. 2-alkylidene-19-nor-vitamin D compounds, havenow been synthesized and tested. Of particular interest are the analogswhich are characterized by the transposition of the ring A exocyclicmethylene group, present in the normal vitamin D skeleton, from carbon10 (C-10) to carbon 2 (C-2), i.e. 2-methylene-19-nor-vitamin Dcompounds. Such vitamin D analogs seemed interesting targets because therelatively small alkylidene (particularly methylene) group at C-2 shouldnot interfere with vitamin D receptor. Moreover, molecular mechanicsstudies performed on the model 1α-hydroxy-2-methylene-19-nor-vitaminsindicate that such molecular modification does not change substantiallythe conformation of the cyclohexanediol ring A. However, introduction ofthe 2-methylene group into 19-nor-vitamin D carbon skeleton changes thecharacter of its 1α- and 3β-A-ring hydroxyls. They are both now in theallylic positions, similarly, as 1α-hydroxyl group (crucial forbiological activity) in the molecule of the natural hormone,1α,25-(OH)₂D₃.

SUMMARY OF THE INVENTION

A class of 1α-hydroxylated vitamin D compounds not known heretofore arethe 19-nor-vitamin D analogs having an alkylidene (particularlymethylene) group at the 2-position, i.e. 2-alkylidene-19-nor-vitamin Dcompounds, particularly 2-methylene-19-nor-vitamin D compounds. Theselatter compounds are those in which the A-ring exocyclic methylene grouptypical of all vitamin D system has been transposed to the carbon 2,i.e. 19-nor-vitamin D analogs having a methylene group at the2-position.

Structurally these novel analogs are characterized by the generalformula I shown below:

where Y₁ and Y₂, which may be the same or different, are each selectedfrom the group consisting of hydrogen and a hydroxy-protecting group, R₆and R₈, which may be the same or different, are each selected from thegroup consisting of hydrogen, alkyl, hydroxyalkyl and fluoroalkyl, or,when taken together represent the group —(CH₂)_(X)— where X is aninteger from 2 to 5, and where the group R represents any of the typicalside chains known for vitamin D type compounds.

More specifically R can represent a saturated or unsaturated hydrocarbonradical of 1 to 35 carbons, that may be straight-chain, branched orcyclic and that may contain one or more additional substituents, such ashydroxy- or protected-hydroxy groups, fluoro, carbonyl, ester, epoxy,amino or other heteroatomic groups. Preferred side chains of this typeare represented by the structure below

where the stereochemical center (corresponding to C-20 in steroidnumbering) may have the R or S configuration, (i.e. either the naturalconfiguration about carbon 20 or the 20-epi configuration), and where Zis selected from Y, —OY, —CH₂OY, —C≡CY, CH═CHY, and —CH₂CH₂CH═CR³R⁴,where the double bond may have the cis or trans geometry, and where Y isselected from hydrogen, methyl, —COR⁵ and a radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR¹ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R², R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an alkylidene group, ═CR²R³,or the group —(CH₂)_(p)—, where p is an integer from 2 to 5, and whereR³ and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, C₁₋₅ alkyl or —OR⁷ where R⁷represents C₁₋₅ alkyl, and wherein any of the CH-groups at positions 20,22, or 23 in the side chain may be replaced by a nitrogen atom, or whereany of the groups —CH(CH₃)—, —CH(R³)—, or —CH(R²)— at positions 20, 22,and 23, respectively, may be replaced by an oxygen or sulfur atom.

The wavy line to the methyl substituent at C-20 indicates that carbon 20may have either the R or S configuration.

Specific important examples of side chains with natural20R-configuration are the structures represented by formulas (a), b),(c), (d) and (e) below. i.e. the side chain as it occurs in25-hydroxyvitamin D₃ (a); vitamin D₃ (b); 25-hydroxyvitamin D₂ (c);vitamin D₂ (d); and the C-24 epimer of 25-hydroxyvitamin D₂ (e):

Specific important examples of side chains with the unnatural 20(S)(also referred to as the 20-epi) configuration are the structuresrepresented by formulas (f), (g), (h), and (i) below:

The above novel compounds exhibit a desired, and highly advantageous,pattern of biological activity. These compounds are characterized byrelatively high intestinal calcium transport activity, as compared tothat of 1α,25-dihydroxyvitamin D₃, while also exhibiting relatively highactivity, as compared to 1α,25-dihydroxyvitamin D₃, in their ability tomobilize calcium from bone. Hence, these compounds are highly specificin their calcemic activity. Their preferential activity on mobilizingcalcium from bone and either high or normal intestinal calcium transportactivity allows the in vivo administration of these compounds for thetreatment of metabolic bone diseases where bone loss is a major concern.Because of their preferential calcemic activity on bone, these compoundswould be preferred therapeutic agents for the treatment of diseaseswhere bone formation is desired, such as osteoporosis, especially lowbone turnover osteoporosis, steroid induced osteoporosis, senileosteoporosis or postmenopausal osteoporosis, as well as osteomalacia andrenal osteodystrophy. The treatment may be transdermal, oral orparenteral. The compounds may be present in a composition in an amountfrom about 0.1 μg/gm to about 50 μg/gm of the composition, and may beadministered in dosages of from about 0.1 μg/day to about 50 μg/day.

The compounds of the invention are also especially suited for treatmentand prophylaxis of human disorders which are characterized by animbalance in the immune system, e.g. in autoimmune diseases, includingmultiple sclerosis, diabetes mellitus, host versus graft reaction, andrejection of transplants; and additionally for the treatment ofinflammatory diseases, such as rheumatoid arthritis and asthma, as wellas the improvement of bone fracture healing and improved bone grafts.Acne, alopecia, skin conditions such as dry skin (lack of dermalhydration), undue skin slackness (insufficient skin firmness),insufficient sebum secretion and wrinkles, and hypertension are otherconditions which may be treated with the compounds of the invention.

The above compounds are also characterized by high cell differentiationactivity. Thus, these compounds also provide therapeutic agents for thetreatment of psoriasis, or as an anti-cancer agent, especially againstleukemia, colon cancer, breast cancer and prostate cancer. The compoundsmay be present in a composition to treat psoriasis in an amount fromabout 0.01 μg/gm to about 100 μg/gm of the composition, and may beadministered topically, transdermally, orally or parenterally in dosagesof from about 0.01 μg/day to about 100 μg/day.

This invention also provides novel intermediate compounds formed duringthe synthesis of the end products. Structurally, these novelintermediates are characterized by the general formulae V, VI, VII,VIII, 1× and X below where Y₁, Y₂, R₆ and R₈ are as previously definedherein.

This invention also provides a novel synthesis for the production of theend products of structure I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the relative activity of2-methylene-19-nor-20(S)-1α,25-dihydroxyvitamin D₃,2-methylene-19-nor-1α,25-dihydroxyvitamin D₃ and 1α,25-dihydroxyvitaminD₃ to compete for binding of [3H]-1,25-(OH)₂-D₃ to the vitamin D pigintestinal nuclear receptor; and

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of2-methylene-19-nor-20(S)-1α,25-dihydroxyvitamin D₃,2-methylene-19-nor-1α,25-dihydroxyvitamin D₃ and 1α,25-dihydroxyvitaminD₃.

DETAILED DESCRIPTION OF THE INVENTION

As used in the description and in the claims, the term“hydroxy-protecting group’ signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.Alkoxyalkyl protecting groups are groupings such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively.

It should be noted in this description that the term “24-homo” refers tothe addition of one methylene group and the term “24-dihomo” refers tothe addition of two methylene groups at the carbon 24 position in theside chain. Likewise, the term “trihomo” refers to the addition of threemethylene groups. Also, the term “26,27-dimethyl” refers to the additionof a methyl group at the carbon 26 and 27 positions so that for exampleR³ and R⁴ are ethyl groups. Likewise, the term “26,27-diethyl” refers tothe addition of an ethyl group at the 26 and 27 positions so that R³ andR⁴ are propyl groups.

In the following lists of compounds, the particular alkylidenesubstituent attached at the carbon 2 position should be added to thenomenclature. For example, if a methylene group is the alkylidenesubstituent, the term “2-methylene” should precede each of the namedcompounds. If an ethylene group is the alkylidene substituent, the term“2-ethylene” should precede each of the named compounds, and so on. Inaddition, if the methyl group attached at the carbon 20 position is inits epi or unnatural configuration, the term “20(S)” or “20-epi” shouldbe included in each of the following named compounds. The namedcompounds could also be of the vitamin D₂ type if desired.

Specific and preferred examples of the 2-alkylidene-compounds ofstructure I when the side chain is unsaturated are:

-   19-nor-24-homo-1,25-dihydroxy-22-dehydrovitamin D₃;-   19-nor-24-dihomo-1,25-dihydroxy-22-dehydrovitamin D₃;-   19-nor-24-trihomo-1,25-dihydroxy-22-dehydrovitamin D₃;-   19-nor-26,27-dimethyl-24-homo-1,25-dihydroxy-22-dehydrovitamin D₃;-   19-nor-26,27-dimethyl-24-dihomo-1,25-dihydroxy-22-dehydrovitamin D₃;-   19-nor-26,27-dimethyl-24-trihomo-1,25-dihydroxy-22-dehydrovitamin    D₃;-   19-nor-26,27-diethyl-24-homo-1,25-dihydroxy-22-dehydrovitamin D₃;-   19-nor-26,27-diethyl-24-dihomo-1,25-dihydroxy-22-dehydrovitamin D₃;-   19-nor-26,27-diethyl-24-trihomo-1,25-dihydroxy-22-dehydrovitamin D₃;-   19-nor-26,27-dipropoyl-24-homo-1,25-dihydroxy-22-dehydrovitamin D₃;-   19-nor-26,27-dipropyl-24-dihomo-1,25-dihydroxy-22-dehydrovitamin D₃;-   19-nor-26,27-dipropyl-24-trihomo-1,25-dihydroxy-22-dehydrovitamin    D₃; and-   19-nor-26,27-dimethylene-1-hydroxy-24-dehydrovitamin D₃.    A particularly preferred side chain unsaturated compound is:-   19-nor-26,27-dimethylene-20(S)-2-methylene-1α-hydroxy-24-dehydrovitamin    D₃.

Specific and preferred examples of the 2-alkylidene-compounds ofstructure I when the side chain is saturated are:

-   19-nor-24-homo-1,25-dihydroxyvitamin D₃;-   19-nor-24-dihomo-1,25-dihydroxyvitamin D₃;-   19-nor-24-trihomo-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-dimethyl-24-homo-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-dimethyl-24-dihomo-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-dimethyl-24-trihomo-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-diethyl-24-homo-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-diethyl-24-dihomo-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-diethyl-24-trihomo-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-dipropyl-24-homo-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-dipropyl-24-dihomo-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-dipropyl-24-trihomo-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-dimethyl-1,25-dihydroxyvitamin D₃;-   19-nor-26,27-dimethylene-1,25-hydroxyvitamin D₃; and-   19-nor-26,27-dimethylene-1-hydroxy-25-methoxyvitamin D₃.    As noted previously, the above saturated side chain compounds should    have the appropriate 2-alkylidene substituent and/or carbon 20    configuration added to the nomenclature. For example, particularly    preferred saturated side chain compounds are:-   19-nor-26,27-dimethyl-20(S)-2-methylene-1α,25-dihydroxyvitamin D₃;    which can also be written as    19-nor-26,27-dihomo-20(S)-2-methylene-1α,25-dihydroxyvitamin D₃;-   19-nor-26,27-dimethylene-20(S)-2-methylene-1α,25-dihydroxyvitamin    D₃; and-   19-nor-26,27-dimethylene-20(S)-2-methylene-1α-hydroxy-25-methoxyvitamin    D₃.

The preparation of 1α-hydroxy-2-alkyl-19-nor-vitamin D compounds,particularly 1α-hydroxy-2-methyl-19-nor-vitamin D compounds, having thebasic structure I can be accomplished by a common general method, i.e.the condensation of a bicyclic Windaus-Grundmann type ketone II with theallylic phosphine oxide III to the corresponding2-methylene-19-nor-vitamin D analogs IV followed by deprotection at C-1and C-3 in the latter compounds:

In the structures II, III, and IV groups Y₁ and Y₂ and R representgroups defined above; Y₁ and Y₂ are preferably hydroxy-protectinggroups, it being also understood that any functionalities in R thatmight be sensitive, or that interfere with the condensation reaction, besuitable protected as is well-known in the art. The process shown aboverepresents an application of the convergent synthesis concept, which hasbeen applied effectively for the preparation of vitamin D compounds[e.g. Lythgoe et al., J. Chem. Soc. Perkin Trans. I, 590 (1978);Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Toh et al., J. Org. Chem. 48,1414 (1983); Baggiolini et al., J. Org. Chem. 51, 3098 (1986); Sardinaet al., J. Org. Chem. 51, 1264 (1986); J. Org. Chem. 51, 1269 (1986);DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca et al., U.S. Pat. No.5,536,713].

Hydrindanones of the general structure II are known, or can be preparedby known methods. Specific important examples of such known bicyclicketones are the structures with the side chains (a), (b), (c) and (d)described above, i.e. 25-hydroxy Grundmann's ketone (f) [Baggiolini etal., J. Org. Chem., 51, 3098 (1986)]; Grundmann's ketone (g) [Inhoffenet al., Chem. Ber. 90, 664 (1957)]; 25-hydroxy Windaus ketone (h)[Baggiolini et al., J. Org. Chem., 51, 3098 (1986)] and Windaus ketone(i) [Windaus et al., Ann., 524, 297 (1936)]:

For the preparation of the required phosphine oxides of generalstructure III, a new synthetic route has been developed starting frommethyl quinicate derivative 1, easily obtained from commercial(1R,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191. The overall process of transformation of the starting methylester 1 into the desired A-ring synthons, is summarized by the SCHEME I.Thus, the secondary 4-hydroxyl group of 1 was oxidized with RuO₄ (acatalytic method with RuCl₃ and NaIO₄ as co-oxidant). Use of such astrong oxidant was necessary for an effective oxidation process of thisvery hindered hydroxyl. However, other more commonly used oxidants canalso be applied (e.g. pyridinium dichromate), although the reactionsusually require much longer time for completion. Second step of thesynthesis comprises the Wittig reaction of the sterically hindered4-keto compound 2 with ylide prepared from methyltriphenylphosphoniumbromide and n-butyllithium. Other bases can be also used for thegeneration of the reactive methylenephosphorane, like t-BuOK, NaNH₂,NaH, K/HMPT, NaN(TMS)₂, etc. For the preparation of the 4-methylenecompound 3 some described modifications of the Wittig process can beused, e.g. reaction of 2 with activated methylenetriphenyl-phosphorane[Corey et al., Tetrahedron Lett. 26, 555 (1985)]. Alternatively, othermethods widely used for methylenation of unreactive ketones can beapplied, e.g. Wittig-Horner reaction with the PO-ylid obtained frommethyldiphenylphosphine oxide upon deprotonation with n-butyllithium[Schosse et al., Chimia 30, 197 (1976)], or reaction of ketone withsodium methylsulfinate [Corey et al., J. Org. Chem. 28, 1128 (1963)] andpotassium methylsulfinate [Greene et al., Tetrahedron Lett. 3755(1976)]. Reduction of the ester 3 with lithium aluminum hydride or othersuitable reducing agent (e.g. DIBALH) provided the diol 4 which wassubsequently oxidized by sodium periodate to the cyclohexanonederivative 5. The next step of the process comprises the Petersonreaction of the ketone 5 with methyl(trimethylsilyl)acetate. Theresulting allylic ester 6 was treated with diisobutylaluminum hydrideand the formed allylic alcohol 7 was in turn transformed to the desiredA-ring phosphine oxide 8. Conversion of 7 to 8 involved 3 steps, namely,in situ tosylation with n-butyllithium and p-toluenesulfonyl chloride,followed by reaction with diphenylphosphine lithium salt and oxidationwith hydrogen peroxide.

Several 2-methylene-19-nor-vitamin D compounds of the general structureIV may be synthesized using the A-ring synthon 8 and the appropriateWindaus-Grundmann ketone II having the desired side chain structure.Thus, for example, Wittig-Horner coupling of the lithium phosphinoxycarbanion generated from 8 and n-butyllithium with the protected25-hydroxy Grundmann's ketone 9 prepared according to publishedprocedure [Sicinski et al., J. Med. Chem. 37, 3730 (1994)] gave theexpected protected vitamin compound 10. This, after deprotection with AG50W-X4 cation exchange resin afforded1α,25-dihydroxy-2-methylene-19-nor-vitamin D₃ (11).

The C-20 epimerization was accomplished by the analogous coupling of thephosphine oxide 8 with protected 20(S)-25-hydroxy Grundmann's ketone 13(SCHEME II) and provided 19-nor-vitamin 14 which after hydrolysis of thehydroxy-protecting groups gave20(S)-1α,25-dihydroxy-2-methylene-19-nor-vitamin D₃ (15).

As noted above, other 2-methylene-19-nor-vitamin D analogs may besynthesized by the method disclosed herein. For example,1α-hydroxy-2-methylene-19-nor-vitamin D₃ can be obtained by providingthe Grundmann's ketone (g).

This invention is described by the following illustrative examples. Inthese examples specific products identified by Arabic numerals (e.g. 1,2, 3, etc) refer to the specific structures so identified in thepreceding description and in the SCHEME I and SCHEME II.

EXAMPLE 1 Preparation of 1α,25-dihydroxy-2-methylene-19-nor-vitamin D₃(11)

Referring first to SCHEME I the starting methyl quinicate derivative 1was obtained from commercial (−)-quinic acid as described previously[Perlman et al., Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al.,U.S. Pat. No. 5,086,191]. 1: mp. 82-82.5° C. (from hexane), ¹H NMR(CDCl₃) 0.098, 0.110, 0.142, and 0.159 (each 3H, each s, 4×SiCH₃), 0.896and 0.911 (9H and 9H, each s, 2×Si-t-Bu), 1.820 (1H, dd, J=13.1, 10.3Hz), 2.02 (1H, ddd, J=14.3, 4.3, 2.4 Hz), 2.09 (1H, dd, J=14.3, 2.8 Hz),2.19 (1H, ddd, J=13.1, 4.4, 2.4 Hz), 2.31 (1H, d, J=2.8 Hz, OH), 3.42(1H, m; after D₂O dd, J=8.6, 2.6 Hz), 3.77 (3H, s), 4.12 (1H, m), 4.37(1H, m), 4.53 (1H, br s, OH).

(a) Oxidation of 4-hydroxy group in methyl quinicate derivative 1.

(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4-oxocyclohexanecarboxylicAcid Methyl Ester (2). To a stirred mixture of ruthenium(III) chloridehydrate (434 mg, 2.1 mmol) and sodium periodate (10.8 g, 50.6 mmol) inwater (42 mL) was added a solution of methyl quinicate 1 (6.09 g, 14mmol) in CCl₄/CH₃CN (1:1, 64 mL). Vigorous stirring was continued for 8h. Few drops of 2-propanol were added, the mixture was poured into waterand extracted with chloroform. The organic extracts were combined,washed with water, dried (MgSO₄) and evaporated to give a dark oilyresidue (ca. 5 g) which was purified by flash chromatography. Elutionwith hexane/ethyl acetate (8:2) gave pure, oily 4-ketone 2 (3.4 g, 56%):¹H NMR (CDCl₃) δ 0.054, 0.091, 0.127, and 0.132 (each 3H, each s,4×SiCH₃), 0.908 and 0.913 (9H and 9H, each s, 2×Si-tBu), 2.22 (1H, dd,J=13.2, 11.7 Hz), 2.28 (1H, dt, J=14.9, 3.6 Hz), 2.37 (1H, dd, J=14.9,3.2 Hz), 2.55 (1H, ddd, J=13.2, 6.4, 3.4 Hz), 3.79 (3H, s), 4.41 (1H, t,J˜3.5 Hz), 4.64 (1H, s, OH), 5.04 (1H, dd, J=11.7, 6.4 Hz); MS m/z(relative intensity) no M⁺, 375 (M⁺-t-Bu, 32), 357 (M⁺-t-Bu —H₂O, 47),243 (31), 225 (57), 73 (100).

(b) Wittig reaction of the 4-ketone 2.

(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4-methylenecyclohexanecarboxylicAcid Methyl Ester (3). To the methyltriphenylphoshonium bromide (2.813g, 7.88 mmol) in anhydrous THF (32 mL) at 0° C. was added dropwisen-BuLi (2.5 M in hexanes, 6.0 mL, 15 mmol) under argon with stirring.Another portion of MePh₃P⁺Br⁻ (2.813 g, 7.88 mmol) was then added andthe solution was stirred at 0° C. for 10 min and at room temperature for40 min. The orange-red mixture was again cooled to 0° C. and a solutionof 4-ketone 2 (1.558 g, 3.6 mmol) in anhydrous THF (16+2 mL) wassyphoned to reaction flask during 20 min. The reaction mixture wasstirred at 0° C. for 1 h and at room temperature for 3 h. The mixturewas then carefully poured into brine cont. 1% HCl and extracted withethyl acetate and benzene. The combined organic extracts were washedwith diluted NaHCO₃ and brine, dried (MgSO₄) and evaporated to give anorange oily residue (ca. 2.6 g) which was purified by flashchromatography. Elution with hexane/ethyl acetate (9:1) gave pure4-methylene compound 3 as a colorless oil (368 mg, 24%): ¹H NMR (CDCl₃)δ 0.078, 0.083, 0.092, and 0.115 (each 3H, each s, 4×SiCH₃), 0.889 and0.920 (9H and 9H, each s, 2×Si-t-Bu), 1.811 (1H, dd, J=12.6, 11.2 Hz),2.10 (2H, m), 2.31 (1H, dd, J=12.6, 5.1 Hz), 3.76 (3H, s), 4.69 (1H, t,J=3.1 Hz), 4.78 (1H, m), 4.96 (2H, m; after D₂O 1H, br s), 5.17 (1H, t,J=1.9 Hz); MS m/z (relative intensity) no M⁺, 373 (M⁺-t-Bu, 57), 355(M⁺-t-Bu —H₂O, 13), 341 (19), 313 (25), 241 (33), 223 (37), 209 (56), 73(100).

(c) Reduction of ester group in the 4-methylene compound 3.

[(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4-methylenecyclohexyl]methanol(4). (i) To a stirred solution of the ester 3 (90 mg, 0.21 mmol) inanhydrous THF (8 mL) lithium aluminum hydride (60 mg, 1.6 mmol) wasadded at 0° C. under argon. The cooling bath was removed after 1 h andthe stirring was continued at 6° C. for 12 h and at room temperature for6 h. The excess of the reagent was decomposed with saturated aq. Na₂SO₄,and the mixture was extracted with ethyl acetate and ether, dried(MgSO₄) and evaporated. Flash chromatography of the residue withhexane/ethyl acetate (9:1) afforded unreacted substrate (12 mg) and apure, crystalline diol 4 (35 mg, 48% based on recovered ester 3): ¹H NMR(CDCl₃+D₂O) δ 0.079, 0.091, 0.100, and 0.121 (each 3H, each s, 4×SiCH₃),0.895 and 0.927 (9H and 9H, each s, 2×Si-t-Bu), 1.339 (1H, t, J˜12 Hz),1.510 (1H, dd, J=14.3, 2.7 Hz), 2.10 (2H, m), 3.29 and 3.40 (1H and 1H,each d, J=11.0 Hz), 4.66 (1H, t, J˜2.8 Hz), 4.78 (1H, m), 4.92 (1H, t,J=1.7 Hz), 5.13 (1H, t, J=2.0 Hz); MS m/z (relative intensity) no M⁺,345 (M⁺-t-Bu, 8), 327 (M⁺-t-Bu —H₂O, 22), 213 (28), 195 (11), 73 (100).

(ii) Diisobutylaluminum hydride (1.5 M in toluene, 2.0 mL, 3 mmol) wasadded to a solution of the ester 3 (215 mg, 0.5 mmol) in anhydrous ether(3 mL) at −78° C. under argon. The mixture was stirred at −78° C. for 3h and at −24° C. for 1.5 h, diluted with ether (10 mL) and quenched bythe slow addition of 2N potassium sodium tartrate. The solution waswarmed to room temperature and stirred for 15 min, then poured intobrine and extracted with ethyl acetate and ether. The organic extractswere combined, washed with diluted (ca. 1%) HCl, and brine, dried(MgSO₄) and evaporated. The crystalline residue was purified by flashchromatography. Elution with hexane/ethyl acetate (9:1) gave crystallinediol 4 (43 mg, 24%).

(d) Cleavage of the vicinal diol 4.

(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-4-methylenecyclohexanone(5). Sodium periodate saturated water (2.2 mL) was added to a solutionof the diol 4 (146 mg, 0.36 mmol) in methanol (9 mL) at 0° C. Thesolution was stirred at 0° C. for 1 h, poured into brine and extractedwith ether and benzene. The organic extracts were combined, washed withbrine, dried (MgSO₄) and evaporated. An oily residue was dissolved inhexane (1 mL) and applied on a silica Sep-Pak cartridge. Pure4-methylenecyclohexanone derivative 5 (110 mg, 82%) was eluted withhexane/ethyl acetate (95:5) as a colorless oil: ¹H NMR (CDCl₃) δ 0.050and 0.069 (6H and 6H, each s, 4×SiCH₃), 0.881 (18H, s, 2×Si-t-Bu), 2.45(2H, ddd, J=14.2, 6.9, 1.4 Hz), 2.64 (2H, ddd, J=14.2, 4.6, 1.4 Hz),4.69 (2H, dd, J=6.9, 4.6 Hz), 5.16 (2H, s); MS m/z (relative intensity)no M⁺, 355 (M⁺-Me, 3), 313 (M⁺-t-Bu, 100), 73 (76).

(e) Preparation of the allylic ester 6.

[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-methylenecyclohexylidene]aceticAcid Methyl Ester (6). To a solution of diisopropylamine (37 μL, 0.28mmol) in anhydrous THF (200 μL) was added n-BuLi (2.5 M in hexanes, 113μL, 0.28 mmol) under argon at −78° C. with stirring, andmethyl(trimethylsilyl)acetate (46 μL, 0.28 mmol) was then added. After15 min, the keto compound 5 (49 mg, 0.132 mmol) in anhydrous THF (200+80μL) was added dropwise. The solution was stirred at −78° C. for 2 h andthe reaction mixture was quenched with saturated NH₄Cl, poured intobrine and extracted with ether and benzene. The combined organicextracts were washed with brine, dried (MgSO₄) and evaporated. Theresidue was dissolved in hexane (1 mL) and applied on a silica Sep-Pakcartridge. Elution with hexane and hexane/ethyl acetate (98:2) gave apure allylic ester 6 (50 mg, 89%) as a colorless oil: ¹H NMR (CDCl₃) δ0.039, 0.064, and 0.076 (6H, 3H, and 3H, each s, 4×SiCH₃), 0.864 and0.884 (9H and 9H, each s, 2×Si-t-Bu), 2.26 (1H, dd, J=12.8, 7.4 Hz),2.47 (1H, dd, J=12.8, 4.2 Hz), 2.98 (1H, dd, J=13.3, 4.0 Hz), 3.06 (1H,dd, J=13.3, 6.6 Hz), 3.69 (3H, s), 4.48 (2H, m), 4.99 (2H, s), 5.74 (1H,s); MS m/z (relative intensity) 426 (M⁺, 2), 411 (M⁺-Me, 4), 369(M⁺-t-Bu, 100), 263 (69).

(f) Reduction of the allylic ester 6.

2-[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-methylenecyclohexylidene]ethanol(7). Diisobutylaluminum hydride (1.5 M in toluene, 1.6 mL, 2.4 mmol) wasslowly added to a stirred solution of the allylic ester 6 (143 mg, 0.33mmol) in toluene/methylene chloride (2:1, 5.7 mL) at −78° C. underargon. Stirring was continued at −78° C. for 1 h and at −46° C.(cyclohexanone/dry ice bath) for 25 min. The mixture was quenched by theslow addition of potassium sodium tartrate (2N, 3 mL), aq. HCl (2N, 3mL) and H₂O (12 mL), and then diluted with methylene chloride (12 mL)and extracted with ether and benzene. The organic extracts werecombined, washed with diluted (ca. 1%) HCl, and brine, dried (MgSO₄) andevaporated. The residue was purified by flash chromatography. Elutionwith hexane/ethyl acetate (9:1) gave crystalline allylic alcohol 7 (130mg, 97%): ¹H NMR (CDCl₃) 0.038, 0.050, and 0.075 (3H, 3H, and 6H, eachs, 4×SiCH₃), 0.876 and 0.904 (9H and 9H, each s, 2×Si-t-Bu), 2.12 (1H,dd, J=12.3, 8.8 Hz), 2.23 (1H, dd, J=13.3, 2.7 Hz), 2.45 (1H, dd,J=12.3, 4.8 Hz), 2.51 (1H, dd, J=13.3, 5.4 Hz), 4.04 (1H, m; after D₂Odd, J=12.0, 7.0 Hz), 4.17 (1H, m; after D₂O dd, J=12.0, 7.4 Hz), 4.38(1H, m), 4.49 (1H, m), 4.95 (1H, br s), 5.05 (1H, t, J=1.7 Hz), 5.69(1H, ˜t, J=7.2 Hz); MS m/z (relative intensity) 398 (M⁺, 2), 383 (M⁺-Me,2), 365 (M⁺-Me—H₂O, 4), 341 (M⁺-t-Bu, 78), 323 (M⁺-t-Bu —H₂O, 10), 73(100).

(g) Conversion of the allylic alcohol 7 into phosphine oxide 8.

[2-[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-methylenecyclohexylidene]ethyl]diphenylphosphineOxide (8). To the allylic alcohol 7 (105 mg, 0.263 mmol) in anhydrousTHF (2.4 mL) was added n-BuLi (2.5 M in hexanes, 105 μL, 0.263 mmol)under argon at 0° C. Freshly recrystallized tosyl chloride (50.4 mg,0.264 mmol) was dissolved in anhydrous THF (480 μL) and added to theallylic alcohol-BuLi solution. The mixture was stirred at 0° C. for 5min and set aside at 0° C. In another dry flask with air replaced byargon, n-BuLi (2.5 M in hexanes, 210 μL, 0.525 mmol) was added to Ph₂PH(93 μL, 0.534 mmol) in anhydrous THF (750 μL) at 0° C. with stirring.The red solution was syphoned under argon pressure to the solution oftosylate until the orange color persisted (ca. ½ of the solution wasadded). The resulting mixture was stirred an additional 30 min at 0° C.,and quenched by addition of H₂O (30 μl). Solvents were evaporated underreduced pressure and the residue was redissolved in methylene chloride(2.4 mL) and stirred with 10% H₂O₂ at 0° C. for 1 h. The organic layerwas separated, washed with cold aq. sodium sulfite and H₂O, dried(MgSO₄) and evaporated. The residue was subjected to flashchromatography. Elution with benzene/ethyl acetate (6:4) gavesemicrystalline phosphine oxide 8 (134 mg, 87%): ¹H NMR (CDCl₃) δ 0.002,0.011, and 0.019 (3H, 3H, and 6H, each s, 4×SiCH₃), 0.855 and 0.860 (9Hand 9H, each s, 2×Si-t-Bu), 2.0-2.1 (3H, br m), 2.34 (1H, m), 3.08 (1H,m), 3.19 (1H, m), 4.34 (2H, m), 4.90 and 4.94 (1H and 1H, each s,), 5.35(1H, q, J=7.4 Hz), 7.46 (4H, m), 7.52 (2H, m), 7.72 (4H, m); MS m/z(relative intensity) no M⁺, 581 (M⁺−1, 1), 567 (M⁺-Me, 3), 525 (M⁺-t-Bu,100), 450 (10), 393 (48).

(h) Wittig-Horner coupling of protected 25-hydroxy Grundrnann's ketone 9with the phosphine oxide 8.

1α,25-Dihydroxy-2-methylene-19-nor-vitamin D₃ (11). To a solution ofphosphine oxide 8 (33.1 mg, 56.8 μmol) in anhydrous THF (450 μL) at 0°C. was slowly added n-BuLi (2.5 M in hexanes, 23 μL, 57.5 μmol) underargon with stirring. The solution turned deep orange. The mixture wascooled to −78° C. and a precooled (−78° C.) solution of protectedhydroxy ketone 9 (9.0 mg, 22.8 μmol), prepared according to publishedprocedure [Sicinski et al., J. Med. Chem. 37, 3730 (1994)], in anhydrousTHF (200+100 μL) was slowly added. The mixture was stirred under argonat −78° C. for 1 h and at 0° C. for 18 h. Ethyl acetate was added, andthe organic phase was washed with brine, dried (MgSO₄) and evaporated.The residue was dissolved in hexane and applied on a silica Sep-Pakcartridge, and washed with hexane/ethyl acetate (99:1, 20 mL) to give19-nor-vitamin derivative 10 (13.5 mg, 78%). The Sep-Pak was then washedwith hexane/ethyl acetate (96:4, 10 mL) to recover some unchangedC,D-ring ketone 9 (2 mg), and with ethyl acetate (10 mL) to recoverdiphenylphosphine oxide (20 mg). For analytical purpose a sample ofprotected vitamin 10 was further purified by HPLC (6.2 mm×25 cmZorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99.9:0.1)solvent system. Pure compound 10 was eluted at R_(V) 26 mL as acolorless oil: UV (in hexane) λ_(max) 244, 253, 263 nm; ¹H NMR (CDCl₃) δ0.025, 0.049, 0.066, and 0.080 (each 3H, each s, 4×SiCH₃), 0.546 (3H, s,18-H₃), 0.565 (6H, q, J=7.9 Hz, 3×SiCH₂), 0.864 and 0.896 (9H and 9H,each s, 2×Si-t-Bu), 0.931 (3H, d, J=6.0 Hz, 21-H₃), 0.947 (9H, t, J=7.9Hz, 3×SiCH₂CH₃), 1.188 (6H, s, 26- and 27-H₃), 2.00 (2H, m), 2.18 (1H,dd, J=12.5, 8.5 Hz, 4β-H), 2.33 (1H, dd, J=13.1, 2.9 Hz, 10β-H), 2.46(1H, dd, J=12.5, 4.5 Hz, 4α-H), 2.52 (1H, dd, J=13.1, 5.8 Hz, 10α-H),2.82 (1H, br d, J=12 Hz, 9β-H), 4.43 (2H, m, 1β- and 3α-H), 4.92 and4.97 (1H and 1H, each s, ═CH₂), 5.84 and 6.22 (1H and 1H, each d, J=11.0Hz, 7- and 6-H); MS m/z (relative intensity) 758 (M⁺, 17), 729 (M⁺-Et,6), 701 (M⁺-t-Bu, 4), 626 (100), 494 (23), 366 (50), 73 (92).

Protected vitamin 10 (4.3 mg) was dissolved in benzene (150 μL) and theresin (AG 50W-X4, 60 mg; prewashed with methanol) in methanol (800 μL)was added. The mixture was stirred at room temperature under argon for17 h, diluted with ethyl acetate/ether (1:1, 4 mL) and decanted. Theresin was washed with ether (8 mL) and the combined organic phaseswashed with brine and saturated NaHCO₃, dried (MgSO₄) and evaporated.The residue was purified by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4mL/min) using hexane/2-propanol (9:1) solvent system. Analytically pure2-methylene-19-nor-vitamin 11 (2.3 mg, 97%) was collected at R_(V) 29 mL(1α,25-dihydroxyvitamin D₃ was eluted at R_(V) 52 mL in the same system)as a white solid: UV (in EtOH) λ_(max) 243.5, 252, 262.5 nm; ¹H NMR(CDCl₃) δ 0.552 (3H, s, 18-H₃), 0.941 (3H, d, J=6.4 Hz, 21-H₃), 1.222(6H, s, 26- and 27-H₃), 2.01 (2H, m), 2.27-2.36 (2H, m), 2.58 (1H, m),2.80-2.88 (2H, m), 4.49 (2H, m, 1β- and 3α-H), 5.10 and 5.11 (1H and 1H,each s, ═CH₂), 5.89 and 6.37 (1H and 1H, each d, J=11.3 Hz, 7- and 6-H);MS m/z (relative intensity) 416 (M⁺, 83), 398 (25), 384 (31), 380 (14),351 (20), 313 (100).

EXAMPLE 2 Preparation of20(S)-1α,25-dihydroxy-2-methylene-19-nor-vitamin D₃ (15)

SCHEME II illustrates the preparation of protected 20(S)-25-hydroxyGrundmann's ketone 13, and its coupling with phosphine oxide 8 (obtainedas described in Example 1).

(a) Silylation of hydroxy ketone 12.

20(S)-25-[(Triethylsilyl)oxy]-des-A,B-cholestan-8-one (13). A solutionof the ketone 12 (Tetrionics, Inc.; 56 mg, 0.2 mmol) and imidazole (65mg, 0.95 mmol) in anhydrous DMF (1.2 mL) was treated with triethylsilylchloride (95 μL, 0.56 mmol), and the mixture was stirred at roomtemperature under argon for 4 h. Ethyl acetate was added and water, andthe organic layer was separated. The ethyl acetate layer was washed withwater and brine, dried (MgSO₄) and evaporated. The residue was passedthrough a silica Sep-Pak cartridge in hexane/ethyl acetate (9:1), andafter evaporation, purified by HPLC (9.4 mm×25 cm Zorbax-Sil column, 4mL/min) using hexane/ethyl acetate (9:1) solvent system. Pure protectedhydroxy ketone 13 (55 mg, 70%) was eluted at R_(V) 35 mL as a colorlessoil: ¹H NMR (CDCl₃) δ 0.566 (6H, q, J=7.9 Hz, 3×SiCH₂), 0.638 (3H, s,18-H₃), 0.859 (3H, d, J=6.0 Hz, 21-H₃), 0.947 (9H, t, J=7.9 Hz,3×SiCH₂CH₃), 1.196 (6H, s, 26- and 27-H₃), 2.45 (1H, dd, J=11.4, 7.5 Hz,14α-H).

(b) Wittig-Horner coupling of protected 20(S)-25-hydroxy Grundmann'sketone 13 with the phosphine oxide 8.

20(S)-1α,25-Dihydroxy-2-methylene-19-nor-vitamin D₃ (15). To a solutionof phosphine oxide 8 (15.8 mg, 27.1 μmol) in anhydrous THF (200 μL) at0° C. was slowly added n-BuLi (2.5 M in hexanes, 11 μL, 27.5 μmol) underargon with stirring. The solution turned deep orange. The mixture wascooled to −78° C. and a precooled (−78° C.) solution of protectedhydroxy ketone 13 (8.0 mg, 20.3 μmol) in anhydrous THF (100 μL) wasslowly added. The mixture was stirred under argon at −78° C. for 1 h andat 0° C. for 18 h. Ethyl acetate was added, and the organic phase waswashed with brine, dried (MgSO₄) and evaporated. The residue wasdissolved in hexane and applied on a silica Sep-Pak cartridge, andwashed with with hexane/ethyl acetate (99.5:0.5, 20 mL) to give19-nor-vitamin derivative 14 (7 mg, 45%) as a colorless oil. The Sep-Pakwas then washed with hexane/ethyl acetate (96:4, 10 mL) to recover someunchanged C,D-ring ketone 13 (4 mg), and with ethyl acetate (10 mL) torecover diphenylphosphine oxide (9 mg). For analytical purpose a sampleof protected vitamin 14 was further purified by HPLC (6.2 mm×25 cmZorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99.9:0.1)solvent system.

14: UV (in hexane) λ_(max) 244, 253.5, 263 nm; ¹H NMR (CDCl₃) δ 0.026,0.049, 0.066, and 0.080 (each 3H, each s, 4×SiCH₃), 0.541 (3H, s,18-H₃), 0.564 (6H, q, J=7.9 Hz, 3×SiCH₂), 0.848 (3H, d, J=6.5 Hz,21-H₃), 0.864 and 0.896 (9H and 9H, each s, 2×Si-t-Bu), 0.945 (9H, t,J=7.9 Hz, 3×SiCH₂CH₃), 1.188 (6H, s, 26- and 27-H₃), 2.15-2.35 (4H, brm), 2.43-2.53 (3H, br m), 2.82 (1H, br d, J=12.9 Hz, 9, —H), 4.42 (2H,m, 1β- and 3α-H), 4.92 and 4.97 (1H and 1H, each s, ═CH₂), 5.84 and 6.22(1H and 1H, each d, J=11.1 Hz, 7- and 6-H); MS m/z (relative intensity)758 (M⁺, 33), 729 (M⁺-Et, 7), 701 (M⁺-t-Bu, 5), 626 (100), 494 (25), 366(52), 75 (82), 73 (69).

Protected vitamin 14 (5.0 mg) was dissolved in benzene (160 μL) and theresin (AG 50W-X4, 70 mg; prewashed with methanol) in methanol (900 μL)was added. The mixture was stirred at room temperature under argon for19 h, diluted with ethyl acetate/ether (1:1, 4 mL) and decanted. Theresin was washed with ether (8 mL) and the combined organic phaseswashed with brine and saturated NaHCO₃, dried (MgSO₄) and evaporated.The residue was purified by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4mL/min) using hexane/2-propanol (9:1) solvent system. Analytically pure2-methylene-19-nor-vitamin 15 (2.6 mg, 95%) was collected at R_(V) 28 mL[(20R)-analog was eluted at R_(V) 29 mL and 1α,25-dihydroxyvitamin D₃ atR_(V) 52 mL in the same system] as a white solid: UV (in EtOH) λ_(max)243.5, 252.5, 262.5 nm; ¹H NMR (CDCl₃) δ 0.551 (3H, s, 18-H₃), 0.858(3H, d, J=6.6 Hz, 21-H₃), 1.215 (6H, s, 26- and 27-H₃), 1.95-2.04 (2H,m), 2.27-2.35 (2H, m), 2.58 (1H, dd, J=13.3, 3.7 Hz), 2.80-2.87 (2H, m),4.49 (2H, m, 1β- and 3α-H), 5.09 and 5.11 (1H and 1H, each s, ═CH₂),5.89 and 6.36 (1H and 1H, each d, J=11.3 Hz, 7- and 6-H); MS m/z(relative intensity) 416 (M⁺, 100), 398 (26), 380 (13), 366 (21), 313(31).

Biological Activity of 2-Methylene-Substituted 19-Nor-1,25-(OH)₂D₃Compounds and their 20(S)-Isomers

The introduction of a methylene group to the 2-position of19-nor-1,25-(OH)₂D₃ or its 20(S)-isomer had little or no effect onbinding to the porcine intestinal vitamin D receptor. All compoundsbound equally well to the porcine receptor including the standard1,25-(OH)₂D₃ (FIG. 1). It might be expected from these results that allof these compounds would have equivalent biological activity.Surprisingly, however, the 2 methylene substitutions produced highlyselective analogs with their primary action on bone. When given for 7days in a chronic mode, the most potent compound tested was the2-methylene-19-nor-20(S)-1,25-(OH)₂D₃ (Table 1). When given at 130pmol/day, its activity on bone calcium mobilization (serum calcium) wasof the order of at least 10 and possible 100-1,000 times more than thatof the native hormone. Under identical conditions, twice the dose of1,25-(OH)₂D₃ gave a serum calcium value of 13.8 mg/100 ml of serumcalcium at the 130 μmol dose. When given at 260 μmol/day, it producedthe astounding value of 14 mg/100 ml of serum calcium at the expense ofbone. To show its selectivity, this compound produced no significantchange in intestinal calcium transport at either the 130 or 260 pmoldose, while 1,25-(OH)₂D₃ produced the expected elevation of intestinalcalcium transport at the only dose tested, i.e. 260 pmol/day. The2-methylene-19-nor-1,25-(OH)₂D₃ also had extremely strong bone calciummobilization at both dose levels but also showed no intestinal calciumtransport activity. The bone calcium mobilization activity of thiscompound is likely to be 10-100 times that of 1,25-(OH)₂D₃. Theseresults illustrate that the 2-methylene and the 20(S)-2-methylenederivatives of 19-nor-1,25-(OH)₂D₃ are selective for the mobilization ofcalcium from bone. Table 2 illustrates the response of both intestineand serum calcium to a single large dose of the various compounds;again, supporting the conclusions derived from Table 1.

The results in FIG. 2 illustrate that2-methylene-19-nor-20(S)-1,25-(OH)₂D₃ is extremely potent in inducingdifferentiation of HL-60 cells to the moncyte. The 2-methylene-19-norcompound had activity similar to 1,25-(OH)₂D₃. These results illustratethe potential of the 2-methylene-19-nor-20(S)-1,25-(OH)₂D₃ and2-methylene-19-nor-1,25-(OH)₂D₃ compounds as anti-cancer agents,especially against leukemia, colon cancer, breast cancer and prostatecancer, or as agents in the treatment of psoriasis.

Competitive binding of the analogs to the porcine intestinal receptorwas carried out by the method described by Dame et al (Biochemistry 25,4523-4534, 1986).

The differentiation of HL-60 promyelocytic into monocytes was determinedas described by Ostrem et al (J. Biol. Chem. 262, 14164-14171, 1987).

TABLE 1 Response of Intestinal Calcium Transport and Serum Calcium (BoneCalcium Mobilization) Activity to Chronic Doses of 2-MethyleneDerivatives of 19-Nor-1,25-(OH)₂D₃ and its 20(S) Isomers Intestinal DoseCalcium (pmol/day/ Transport Serum Calcium Group 7 days) (S/M) (mg/100ml) Vitamin D Deficient Vehicle 5.5 ± 0.2  5.1 ± 0.16 1,25-(OH)₂D₃Treated 260 6.2 ± 0.4 7.2 ± 0.5 2-Methylene-19-Nor-1,25- 130 5.3 ± 0.49.9 ± 0.2 (OH)₂D₃ 260 4.9 ± 0.6 9.6 ± 0.3 2-Methylene-19-Nor-20(S)- 1305.7 ± 0.8 13.8 ± 0.5  1,25-(OH)₂D₃ 260 4.6 ± 0.7 14.4 ± 0.6 

Male weanling rats were obtained from Sprague Dawley Co. (Indianapolis,Ind.) and fed a 0.47% calcium, 0.3% phosphorus vitamin D-deficient dietfor 1 week and then given the same diet containing 0.02% calcium, 0.3%phosphorus for 2 weeks. During the last week they were given theindicated dose of compound by intraperitoneal injection in 0.1 ml 95%propylene glycol and 5% ethanol each day for 7 days. The control animalsreceived only the 0.1 ml of 95% propylene glycol, 5% ethanol.Twenty-four hours after the last dose, the rats were sacrificed andintestinal calcium transport was determined by everted sac technique aspreviously described and serum calcium determined by atomic absorptionspectrometry on a model 3110 Perkin Elmer instrument (Norwalk, Conn.).There were 5 rats per group and the values represent mean±SEM.

TABLE 2 Response of Intestinal Calcium Transport and Serum Calcium (BoneCalcium Mobilization) Activity to a Single Dose of the2-Methylene-Derivatives of 19-Nor-1,25-(OH)₂D₃ and its 20(S) IsomersIntestinal Calcium Transport Serum Calcium Group (S/M) (mg/100 ml) -DControl 4.2 ± 0.3 4.7 ± 0.1 1,25-(OH)₂D₃ 5.8 ± 0.3 5.7 ± 0.22-Methylene-19-Nor-1,25-(OH)₂D₃ 5.3 ± 0.5 6.4 ± 0.12-Methylene-19-Nor-20(S)-1,25-(OH)₂D₃ 5.5 ± 0.6 8.0 ± 0.1

Male Holtzman strain weanling rats were obtained from the Sprague DawleyCo. (Indianapolis, Ind.) and fed the 0.47% calcium, 0.3% phosphorus dietdescribed by Suda et al. (J. Nutr. 100, 1049-1052, 1970) for 1 week andthen fed the same diet containing 0.02% calcium and 0.3% phosphorus for2 additional weeks. At this point, they received a single intrajugularinjection of the indicated dose dissolved in 0.1 ml of 95% propyleneglycol/5% ethanol. Twenty-four hours later they were sacrificed andintestinal calcium transport and serum calcium were determined asdescribed in Table 1. The dose of the compounds was 650 μmol and therewere 5 animals per group. The data are expressed as mean±SEM.

EXAMPLE 3 Preparation of20(S)-1α,25-Dihydroxy-2-methylene-26,27-dihomo-19-norvitamin D₃ (35).Reference is made to Scheme III

20(S)-25-[(Triethylsilyl)oxy]-des-A,B-26,27-dihomocholestan-8-one (32).To a solution of 20(S)-25-hydroxy Grundmann's ketone analog 31(Tetrionics, Madison, Wis.; 18.5 mg, 0.06 mmol) in anhydrous CH₂Cl₂ (60μL) was added 2,6-lutidine (17.4 μL, 0.15 mmol) and triethylsilyltrifluoromethanesulfonate (20.3 μL, 0.09 mmol). The mixture was stirredat room temperature under argon for 1 h. Benzene was added and water,and the organic layer was separated, washed with sat. CuSO₄ and water,dried (MgSO₄) and evaporated. The oily residue was redissolved in hexaneand applied on a silica Sep-Pak cartridge (2 g). Elution with hexane (10mL) gave a small quantity of less polar compounds; further elution withhexane/ethyl acetate (9:1) provided the silylated ketone. Finalpurification was achieved by HPLC (10-mm×25-cm Zorbax-Sil column, 4mL/min) using hexane/ethyl acetate (95:5) solvent system. Pure protectedhydroxy ketone 32 (16.7 mg, 66%) was eluted at R_(V) 37 mL as acolorless oil: ¹H NMR (CDCl₃) 0.573 (6H, q, J=7.9 Hz, 3×SiCH₂), 0.639(3H, s, 18-H₃), 0.825 (6H, t, J=7.5 Hz, 26- and 27-CH₃), 0.861 (3H, d,J=6.1 Hz, 21-H₃), 0.949 (9H, t, J=7.9 Hz, 3×SiCH₂CH₃), 2.45 (1H, dd,J=11.4, 7.6 Hz, 14α-H).

20(S)-1α,25-Dihydroxy-2-methylene-26,27-dihomo-19-norvitamin D₃ (35). Toa solution of phosphine oxide 33 (9.1 mg, 15.6 μmol) in anhydrous THF(150 μL) at 0° C. was slowly added n-BuLi (2.5 M in hexanes, 7 μL, 17.5μmol) under argon with stirring. The solution turned deep orange. It wasstirred for 10 min at 0° C., then cooled to −78° C. and a precooled(−78° C.) solution of protected hydroxy ketone 32 (16.5 mg, 39.0 μmol)in anhydrous THF (300+100 μL) was slowly added. The mixture was stirredunder argon at −78° C. for 1.5 h and at 0° C. for 19 h. Water and ethylacetate were added, and the organic phase was washed with brine, dried(MgSO₄) and evaporated. The residue was dissolved in hexane and appliedon a silica Sep-Pak cartridge, and washed with hexane/ethyl acetate(99.7:0.3, 20 mL) to give slightly impure 19-norvitamin derivative 34(ca. 4 mg). The Sep-Pak was then washed with hexane/ethyl acetate (96:4,10 mL) to recover some unchanged C,D-ring ketone (contaminated with14β-isomer), and with ethyl acetate (10 mL) to recover diphenylphosphineoxide 33 (ca. 6 mg) that was subsequently purified by HPLC (10-mm×25-cmZorbax-Sil column, 4 mL/min) using hexane/2-propanol (9:1) solventsystem; pure compound 33 (5.1 mg) was eluted at R_(V) 36 mL. Theprotected vitamin 34 was further purified by HPLC (6.2-mm×25-cmZorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99.9:0.1)solvent system. Pure compound 34 (3.6 mg, 67% yield considering therecovery of unreacted 33) was eluted at R_(V) 19 mL as a colorless oil:UV (in hexane) max 244.0, 252.5, 262.5 nm; ¹H NMR (CDCl₃) 0.026, 0.048,0.066, and 0.079 (each 3H, each s, 4×SiCH₃), 0.544 (3H, s, 18-H₃), 0.570(6H, q, J=7.9 Hz, 3×SiCH₂), 0.821 (6H, t, J=7.5 Hz, 26- and 27-CH₃),0.849 (3H, d, J=6.7 Hz, 21-H₃), 0.864 and 0.896 (9H and 9H, each s,2×Si-t-Bu), 0.946 (9H, t, J=7.9 Hz, 3×SiCH₂CH₃), 1.99 (2H, m), 2.18 (1H,dd, J=12.6, 8.2 Hz, 4, —H), 2.34 (1H, dd, J=13.0, 2.9 Hz, 10, —H), 2.46(1H, dd, J=12.6, 4.3 Hz, 4-H), 2.51 (1H, dd, J=13.0, 6.2 Hz, 10-H), 2.82(1H, br d, J=12 Hz, 9β-H), 4.43 (2H, m, 1β- and 3-H), 4.92 and 4.97 (1Hand 1H, each s, ═CH₂), 5.84 and 6.22 (1H and 1H, each d, J=11.2 Hz, 7-and 6-H); MS m/z (relative intensity) 786 (M⁺, 15), 757 (M⁺-Et, 22), 729(M⁺-t-Bu, 5), 654 (100), 522 (15), 366 (43), 201 (31).

Protected vitamin 34 (3.5 mg) was dissolved in benzene (150 μL) and theresin (AG 50W-X4, 40 mg; prewashed with methanol) in methanol (550 μL)was added. The mixture was stirred at room temperature under argon for14 h, diluted with ethyl acetate/ether (1:1, 4 mL) and decanted. Theresin was washed with ether (8 mL) and the combined organic phaseswashed with brine and saturated NaHCO₃, dried (MgSO₄) and evaporated.The residue was purified by HPLC (6.2-mm×25-cm Zorbax-Sil column, 4mL/min) using hexane/2-propanol (9:1) solvent system. Analytically pure2-methylene-19-norvitamin 35 (1.22 mg, 62%) was collected at R_(V) 21 mLas a white solid: UV (in EtOH) λ_(max) 243.5, 252.0, 262.0 nm; ¹H NMR(CDCl₃) δ 0.550 (3H, s, 18-H₃), 0.855 (3H, d, J=6.8 Hz, 21-H₃), 0.860(6H, t, J=7.5 Hz, 26- and 27-CH₃), 2.00 (3H, m), 2.30 (1H, dd, J=13.3,8.6 Hz, 10α-H), 2.33 (1H, dd, J=13.3, 6.3 Hz, 4β-H), 2.58 (1H, dd,J=13.3, 3.9 Hz, 4α-H), 2.82 (1H, br d, J=12 Hz, 9β-H), 2.85 (1H, dd,J=13.3, 4.7 Hz, 10β-H), 4.48 (2H, m, 1β- and 3α-H), 5.09 and 5.11 (1Hand 1H, each s, ═CH₂), 5.89 and 6.36 (1H and 1H, each d, J=11.3 Hz, 7-and 6-H); MS m/z (relative intensity) 444 (M⁺, 100), 426 (35), 408 (11),397 (19), 379 (32), 341 (31), 287 (32), 273 (43), 269 (28), 251 (22);exact mass calcd for C₂₉H₄₈O₃ 444.3603, found 444.3602.

Biological Activity of20(S)-1α,25-dihydroxy-2-methylene-26,27-dihomo-19-norvitamin D₃ (35)

Competitive binding of the analogs to the porcine intestinal receptorwas carried out by the method described by Dame et al (Biochemistry 25,4523-4534, 1986).

The differentiation of HL-60 promyleocytic into monocytes was determinedas described by Ostrem et al (J. Biol. Chem. 262, 14164-14171, 1987).

TABLE 3 VDR Binding Properties^(a) and HL-60 DifferentiatingActivities^(b) of 2-Substituted Analogs of20(S)-1α,25-Dihydroxy-26,27-dihomo-19-norvitamin D₃ VDR Binding HL-60Differentiation Compd. Binding Activity Compound no. ED₅₀ (M) ratio ED₅₀(M) ratio 1α,25-(OH)₂D₃ 8.7 × 10⁻¹⁰ 1 4.0 × 10⁻⁹ 1 2-methylene- 35 4.3 ×10⁻⁹ 4.9 2.6 × 10⁻¹¹ 0.01 26,27-dihomo- 19-nor-20(S)- 1α,25-(OH)₂D₃^(a)Competitive binding of 1α,25-(OH)₂D₃ and the synthesized vitamin Danalogs to the porcine intestinal vitamin D receptor. The experimentswere carried out in triplicate on two different occasions. The ED₅₀values are derived from dose-response curves and represent the analogconcentration required for 50% displacement of the radiolabeled1α,25-(OH)₂D₃ from the receptor protein. Binding ratio is the ratio ofthe analog average ED₅₀ to the ED₅₀ for 1α,25-(OH)₂D₃. ^(b)Induction ofdifferentiation of HL-60 promyelocytes to monocytes by 1α,25-(OH)₂D₃ andthe synthesized vitamin D analogs. Differentiation state was determinedby measuring the percentage of cells reducing nitro blue tetrazolium(NBT). The experiment was repeated three times. The values ED₅₀ arederived from dose-response curves and represent the analog concentrationcapable of inducing 50% maturation. Differentiation activity radio isthe ratio of the analog average ED₅₀ to the ED₅₀ for 1α,25-(OH)₂D₃.

TABLE 4 Support of Intestinal Calcium Transport and Bone CalciumMobilization by 2-Substituted Analogs of of20(S)-1α,25-Dihydroxy-26,27-dihomo-19-norvitamin D₃ in VitaminD-Deficient Rats on a Low-Calcium Diet^(a) Compd. Amount Ca TransportS/M Serum Ca Compound no. (pmol) (mean ± SEM) (mean ± SEM) none(control) 0 2.7 ± 0.3^(b) 4.7 ± 0.2^(b) 1α,25-(OH)₂D₃ 260 7.2 ± 0.6^(c)5.6 ± 0.2^(c) 2-methylene-26,27-dihomo- 35 15 4.0 ± 0.4^(d) ¹ 5.3 ±0.1^(d) ¹ 19-nor-20(S)-1α,25-(OH)₂D₃ 32 8.2 ± 0.6^(d) ² 7.3 ± 0.4^(d) ²^(a)Weanling male rats were maintained on a 0.47% Ca diet for 1 week andthen switched to a low-calcium diet containing 0.02% Ca for anadditional 3 weeks. During the last week, they were dosed daily with theappropriate vitamin D compound for 7 consecutive days. All doses wereadministered intraperitoneally in 0.1 ml propylene glycol/ethanol(95:5). Controls received the vehicle. Determinations were made 24 hafter the last dose. There were at least 6 rats per group. Statisticalanalysis was done by Student's t-test. Statistical data: serosal/mucosal(S/M), ^(b)from ^(c) and ^(d) ² , p < 0.001, ^(b) from ^(d) ¹ , NS;serum calcium, ^(b) from ^(c), p < 0.05, ^(b) from ^(d) ¹ , NS, ^(b)from ^(d) ² , p = 0.005.

EXAMPLE 4 Preparation of20(S)-26,27-dimethylene-1α-hydroxy-2-methylene-24-dehydro-19-norvitaminD₃ (45);20(S)-26,27-dimethylene-1α-hydroxy-25-methoxy-2-methylene-19-norvitaminD₃ (46); and20(S)-1α,25-dihydroxy-26,27-dimethylene-2-methylene-19-norvitamin D₃(47)

Reference is made to SCHEME IV.

20(S)-25-[(Triethylsilyl)oxy]-des-A,B-26,27-dimethylene-cholestan-8-one(42). To a solution of 20(S)-25-hydroxy Grundmann's ketone analog 41(Tetrionics, Madison, Wis.; 15.0 mg, 0.049 mmol) in anhydrous CH₂Cl₂ (50μL) was added 2,6-lutidine (15 μL, 0.129 mmol) and triethylsilyltrifluoromethanesulfonate (17.0 μL, 0.075 mmol). The mixture was stirredat room temperature under argon for 1 h. Benzene was added and water,and the organic layer was separated, washed with sat. CuSO₄ and water,dried (MgSO₄) and evaporated. The oily residue was redissolved in hexaneand applied on a silica Sep-Pak cartridge (2 g). Elution with hexane (10mL) gave a small quantity of less polar compounds; further elution withhexane/ethyl acetate (9:1) provided the silylated ketone. Finalpurification was achieved by HPLC (10-mm×25-cm Zorbax-Sil column, 4mL/min) using hexane/ethyl acetate (95:5) solvent system. Pure protectedhydroxy ketone 42 (9.4 mg, 46%) was eluted at R_(V) 39 mL as a colorlessoil: ¹H NMR (CDCl₃) 0.576 (6H, q, J=7.9 Hz, 3×SiCH₂), 0.638 (3H, s,18-H₃), 0.865 (3H, d, J=6.1 Hz, 21-H₃), 0.949 (9H, t, J=7.9 Hz,3×SiCH₂CH₃), 2.45 (1H, dd, J=11.4, 7.5 Hz, 14α-H).

20(S)-1α,25-Dihydroxy-26,27-dimethylene-2-methylene-19-norvitamin D₃(47). To a solution of phosphine oxide 43 (17.7 mg, 30.4 μmol) inanhydrous THF (300 μL) at 0° C. was slowly added n-BuLi (2.5 M inhexanes, 13 μL, 32.5 μmol) under argon with stirring. The solutionturned deep orange. It was stirred for 10 min at 0° C., then cooled to−78° C. and a precooled (−78° C.) solution of protected hydroxy ketone41 (17.8 mg, 42.3 μmol) in anhydrous THF (300+100 μL) was slowly added.The mixture was stirred under argon at −78° C. for 1.5 h and at 0° C.for 18 h. Water and ethyl acetate were added, and the organic phase waswashed with brine, dried (MgSO₄) and evaporated. The residue wasdissolved in hexane and applied on a silica Sep-Pak cartridge, andwashed with hexane/ethyl acetate (99.7:0.3, 20 mL) to give slightlyimpure 19-norvitamin derivative 44 (ca. 11 mg). The Sep-Pak was thenwashed with hexane/ethyl acetate (96:4, 10 mL) to recover some unchangedC,D-ring ketone (contaminated with 14β-isomer), and with ethyl acetate(10 mL) to recover diphenylphosphine oxide 43 (ca. 8 mg) that wassubsequently purified by HPLC (10-mm×25-cm Zorbax-Sil column, 4 mL/min)using hexane/2-propanol (9:1) solvent system; pure compound 43 (7.6 mg)was eluted at R_(V) 36 mL. The protected vitamin 44 was further purifiedby HPLC (6.2-mm×25-cm Zorbax-Sil column, 4 mL/min) using hexane/ethylacetate (99.9:0.1) solvent system. Pure compound 44 (10.1 mg, 74% yieldconsidering the recovery of unreacted 43) was eluted at R_(V) 27 mL as acolorless oil: UV (in hexane) max 244.0, 252.5, 262.5 nm; ¹H NMR (CDCl₃)δ 0.027, 0.048, 0.067, and 0.080 (each 3H, each s, 4×SiCH₃), 0.544 (3H,s, 18-H₃), 0.575 (6H, q, J=7.9 Hz, 3×SiCH₂), 0.854 (3H, d, J=6.1 Hz,21-H₃), 0.866 and 0.896 (9H and 9H, each s, 2×Si-t-Bu), 0.947 (9H, t,J=7.9 Hz, 3×SiCH₂CH₃), 1.99 (2H, m), 2.18 (1H, dd, J=12.8, 8.6 Hz,4β-H), 2.34 (1H, dd, J=13.2, 2.7 Hz, 10β-H), 2.46 (1H, dd, J=12.8, 4.4Hz, 4α-H), 2.51 (1H, dd, J=13.2, 6.0 Hz, 10α-H), 2.82 (1H, br d, J=12Hz, 9β-H), 4.42 (2H, m, 1β- and 3α-H), 4.92 and 4.97 (1H and 1H, each s,═CH₂), 5.84 and 6.22 (1H and 1H, each d, J=11.2 Hz, 7- and 6-H); MS m/z(relative intensity) 784 (M⁺, 8), 755 (M⁺-Et, 4), 727 (M⁺-t-Bu, 6), 652(100), 520 (31), 366 (49), 199 (23).

Protected vitamin 44 (7.0 mg) was dissolved in benzene (220 μL) and theresin (AG 50W-X4, 95 mg; prewashed with methanol) in methanol (1.2 mL)was added. The mixture was stirred at room temperature under argon for21 h, diluted with ethyl acetate/ether (1:1, 4 mL) and decanted. Theresin was washed with ether (10 mL) and the combined organic phaseswashed with brine and saturated NaHCO₃, dried (MgSO₄) and evaporated.The residue was separated by HPLC (6.2-mm×25-cm Zorbax-Sil column, 4mL/min) using hexane/2-propanol (9:1) solvent system and the followinganalytically pure 2-methylene-19-norvitamins were isolated:11α-hydroxy-25-dehydrovitamin 45 (0.68 mg, 17%) was collected at R_(V)13 mL, 1α-hydroxy-25-methoxyvitamin 46 (0.76 mg, 19%) was collected atR_(V) 16 mL and 1α,25-dihydroxyvitamin 47 (2.0 mg, 51%) was collected atR_(V) 21 mL.

45: UV (in EtOH) λ_(max) 243.5, 251.5, 262.0 nm; ¹H NMR (CDCl₃) δ 0.542(3H, s, 18-H₃), 0.847 (3H, d, J=6.5 Hz, 21-H₃), 1.93-2.07 (4H, m),2.18-2.25 (2H, m), 2.26-2.36 (4H, m), 2.58 (1H, dd, J=13.3, 3.9 Hz,4α-H), 2.82 (1H, br d, J=13 Hz, 9β-H), 2.85 (1H, dd, J=13.3, 4.5 Hz,10-H), 4.48 (2H, m, 1β- and 3α-H), 5.09 and 5.11 (1H and 1H, each s,═CH₂), 5.32 (1H, m, w/2=7 Hz, 24-H), 5.88 and 6.36 (1H and 1H, each d,J=11.1 Hz, 7- and 6-H); MS m/z (relative intensity) 424 (M⁺, 100), 406(7), 339 (16), 287 (16), 271 (24), 269 (17), 251 (12); exact mass calcdfor C₂₉H₄₄O₂ 424.3341, found 424.3343.

46: UV (in EtOH) λ_(max) 243.5, 252.0, 262.0 nm; ¹H NMR (CDCl₃) δ 0.553(3H, s, 18-H₃), 0.858 (3H, d, J=6.5 Hz, 21-H₃), 1.95-2.05 (2H, m), 2.30(1H, dd, J=13.3, 8.3 Hz, 10α-H), 2.33 (1H, dd, J=13.4, 6.0 Hz, 4β-H),2.58 (1H, dd, J=13.4, 3.8 Hz, 4α-H), 2.82 (1H, br d, J=13 Hz, 9β-H),2.85 (1H, dd, J=13.3, 4.4 Hz, 10β-H), 3.13 (3H, s, OCH₃), 4.48 (2H, m,1β- and 3α-H), 5.09 and 5.11 (1H and 1H, each s, ═CH₂), 5.89 and 6.36(1H and 1H, each d, J=11.2 Hz, 7- and 6-H); MS m/z (relative intensity)456 (M⁺, 54), 424 (27), 406 (12), 339 (16), 287 (13), 271 (41), 99(100); exact mass calcd for C₃₀H₄₈O₃ 456.3603, found 456.3603.

47: UV (in EtOH) max 243.5, 252.0, 262.0 nm; ¹H NMR (CDCl₃) δ 0.551 (3H,s, 18-H₃), 0.859 (3H, d, J=6.6 Hz, 21-H₃), 1.95-2.05 (2H, m), 2.30 (1H,dd, J=13.5, 8.4 Hz, 10α-H), 2.33 (1H, dd, J=13.3, 6.3 Hz, 4β-H), 2.58(1H, dd, J=13.3, 4.0 Hz, 4α-H), 2.82 (1H, br d, J=12 Hz, 9β-H), 2.85(1H, dd, J=13.5, 4.4 Hz, 10β-H), 4.48 (2H, m, 1β- and 3α-H), 5.09 and5.11 (1H and 1H, each s, ═CH₂), 5.89 and 6.36 (1H and 1H, each d, J=11.3Hz, 7- and 6-H); MS m/z (relative intensity) 442 (M⁺, 100), 424 (47),406 (15), 339 (34), 287 (27), 271 (42), 269 (36), 251 (26); exact masscalcd for C₂₉H₄₆O₃ 442.3447, found 442.3442.

Biological Activity of20(S)-26,27-dimethylene-1α-hydroxy-2-methylene-24-dehydro-19-norvitaminD₃ (45);20(S)-26,27-dimethylene-1α-hydroxy-25-methoxy-2-methylene-19-norvitaminD₃ (46); and20(S)-1α,25-dihydroxy-26,27-dimethylene-2-methylene-19-norvitamin D₃(47)

Competitive binding of the analogs to the porcine intestinal receptorwas carried out by the method described by Dame et al (Biochemistry 25,4523-4534, 1986).

The differentiation of HL-60 promyelocytic into monocytes was determinedas described by Ostrem et al (J. Biol. Chem. 262, 14164-14171, 1987).

TABLE 5 VDR Binding Properties^(a) and HL-60 DifferentiatingActivities^(b) of Side Chain Analogs of 20(S)-26,27-dimethylene-1α-hydroxy-2-methylene-19-norvitamin D₃ VDR Binding HL-60Differentiation Compd. Binding Activity Compound no. ED₅₀ (M) ratio ED₅₀(M) ratio 1α,25- 8.7 × 10⁻¹⁰ 1 4.0 × 10⁻⁹ 1 (OH)₂D₃ 26,27-dimeth- 45 2.9× 10⁻⁸ 33 4.1 × 10⁻⁹ 1.0 ylene-2-meth- ylene 24-de- hydro-19-nor-20(S)-1α- OH-D₃ 26,27-dimeth- 46 1.5 × 10⁻⁸ 17 4.3 × 10⁻⁹ 1.1ylene-2-meth- ylene 25-meth- oxy-19-nor- 20(S)-1α- OH-D₃ 26,27-dimeth-47 2.7 × 10⁻⁹ 3.1 3.6 × 10⁻¹¹ 0.01 ylene-2-meth- ylene-19- or-20(S)-1α,25- (OH)₂D₃ ^(a)Competitive binding of 1α,25-(OH)₂D₃ and thesynthesized vitamin D analogs to the porcine intestinal vitamin Dreceptor. The experiments were carried out in triplicate on twodifferent occasions. The ED₅₀ values are derived from dose-responsecurves and represent the analog concentration required for 50%displacement of the radiolabeled 1α,25-(OH)₂D₃ from the receptorprotein. Binding ratio is the ratio of the analog average ED₅₀ to theED₅₀ for 1α,25-(OH)₂D₃. ^(b)Induction of differentiation of HL-60promyelocytes to monocytes by 1α,25-(OH)₂D₃ and the synthesized vitaminD analogs. Differentiation state was determined by measuring thepercentage of cells reducing nitro blue tetrazolium (NBT). Theexperiment was repeated three times. The values ED₅₀ are derived fromdose-response curves and represent the analog concentration capable ofinducing 50% maturation. Differentiation activity radio is the ratio ofthe analog average ED₅₀ to the ED₅₀ for 1α,25-(OH)₂D₃.

TABLE 6 Support of Intestinal Calcium Transport and Bone CalciumMobilization by Side Chain Analogs at20(S)-26,27-dimethylene-1α-hydroxy-2-methylene-19- norvitamin D₃ inVitamin D-Deficient Rats on a Low-Calcium Diet^(a) Compd. Amount CaTransport S/M Serum Ca Compound no. (pmol) (mean ± SEM) (mean ± SEM)none (control) 0 2.7 ± 0.3^(b)  4.7 ± 0.2^(b) 1α,25-(OH)₂D₃ 260 7.2 ±0.6^(c)  5.6 ± 0.2^(c) 26,27-dimethylene-2-methylene- 47 15 5.6 ±0.6^(d) ¹  5.4 ± 0.2^(d) ¹ 19-nor-20(S)-1α,25-(OH)₂D₃ 32 5.3 ± 0.5^(d) ² 6.4 ± 0.2^(d) ² none (control) 0 3.6 ± 0.4^(b)  5.0 ± 0.1^(b)1α,25-(OH)₂D₃ 260 5.0 ± 0.4^(c)  6.3 ± 0.2^(c)26,27-dimethylene-2-methylene- 45 65 5.5 ± 0.8^(d) ¹  5.7 ± 0.1^(d) ¹24-dehydro-19-nor-20(S)-1α-OH-D₃ 260 4.3 ± 0.5^(d) ² 10.8 ± 0.3^(d) ²26,27-dimethylene-2-methylene- 46 65 5.5 ± 0.8^(e) ¹  5.7 ± 0.1^(e) ¹25-methoxy-19-nor-20(S)-1α-OH-D₃ 260 4.3 ± 0.5^(e) ² 10.8 ± 0.3^(e) ²^(a)Weanling male rats were maintained on a 0.47% Ca diet for 1 week andthen switched to a low-calcium diet containing 0.02% Ca for anadditional 3 weeks. During the last week, they were dosed daily with theappropriate vitamin D compound for 7 consecutive days. All doses wereadministered intraperitoneally in 0.1 ml propylene glycol/ethanol(95:5). Controls received the vehicle. Determinations were made 24 hafter the last dose. There were at least 6 rats per group. Statisticalanalysis was done by Student's t-test. Statistical data: serosal/mucosal(S/M), panel 1, ^(b)from ^(c), p < 0.001, ^(b) from ^(d) ¹ and ^(d) ² ,p = 0.001; panel 2, ^(b) from ^(c) and ^(e) ¹ , p < 0.05, ^(b) from ^(d)¹ , ^(d) ² , and ^(e) ² , NS; serum calcium, panel 1, ^(b) from ^(c), p< 0.05, ^(b) from ^(d) ¹ , NS, ^(b) from ^(d) ² , p = 0.005; panel 2,^(b) from ^(c), p < 0.01, ^(b) from ^(d) ¹ , NS, ^(b) from ^(d) ² and^(e) ¹ , p = 0.05, ^(b) from ^(e) ² , p < 0.001.

For treatment purposes, the novel compounds of this invention defined byformula I may be formulated for pharmaceutical applications as asolution in innocuous solvents, or as an emulsion, suspension ordispersion in suitable solvents or carriers, or as pills, tablets orcapsules, together with solid carriers, according to conventionalmethods known in the art. Any such formulations may also contain otherpharmaceutically-acceptable and non-toxic excipients such asstabilizers, anti-oxidants, binders, coloring agents or emulsifying ortaste-modifying agents.

The compounds may be administered orally, topically, parenterally ortransdermally. The compounds are advantageously administered byinjection or by intravenous infusion or suitable sterile solutions, orin the form of liquid or solid doses via the alimentary canal, or in theform of creams, ointments, patches, or similar vehicles suitable fortransdermal applications. Doses of from 0.1 μg to 50 μg per day of thecompounds are appropriate for treatment purposes, such doses beingadjusted according to the disease to be treated, its severity and theresponse of the subject as is well understood in the art. Since the newcompounds exhibit specificity of action, each may be suitablyadministered alone, or together with graded doses of another activevitamin D compound—e.g. 1α-hydroxyvitamin D₂ or D₃, or1α,25-dihydroxyvitamin D₃—in situations where different degrees of bonemineral mobilization and calcium transport stimulation is found to beadvantageous.

Compositions for use in the above-mentioned treatment of psoriasis andother malignancies comprise an effective amount of one or more2-alkylidene-19-nor-vitamin D compound as defined by the above formula Ias the active ingredient, and a suitable carrier. An effective amount ofsuch compounds for use in accordance with this invention is from about0.01 μg to about 100 μg per gm of composition, and may be administeredtopically, transdermally, orally or parenterally in dosages of fromabout 0.1 μg/day to about 100 μg/day.

The compounds may be formulated as creams, lotions, ointments, topicalpatches, pills, capsules or tablets, or in liquid form as solutions,emulsions, dispersions, or suspensions in pharmaceutically innocuous andacceptable solvent or oils, and such preparations may contain inaddition other pharmaceutically innocuous or beneficial components, suchas stabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds are advantageously administered in amounts sufficient toeffect the differentiation of promyelocytes to normal macrophages.Dosages as described above are suitable, it being understood that theamounts given are to be adjusted in accordance with the severity of thedisease, and the condition and response of the subject as is wellunderstood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For asthma treatment, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

In its broadest application, the present invention relates to any19-nor-2-alkylidene analogs of vitamin D which have the vitamin Dnucleus. By vitamin D nucleus, it is meant a central part consisting ofa substituted chain of five carbon atoms which correspond to positions8, 14, 13, 17 and 20 of vitamin D, and at the ends of which areconnected at position 20 a structural moiety representing any of thetypical side chains known for vitamin D type compounds (such as R aspreviously defined herein), and at position 8 the 5,7-diene moietyconnected to the A-ring of an active 1-hydroxy vitamin D analog (asillustrated by formula I herein). Thus, various known modifications tothe six-membered C-ring and the five-membered D-ring typically presentin vitamin D, such as the lack of one or the other or both, are alsoembraced by the present invention.

Accordingly, compounds of the following formulae Ia, are along withthose of formula I, also encompassed by the present invention:

In the above formula Ia, the definitions of Y₁, Y₂, R₆, R₈ and Z are aspreviously set forth herein. With respect to X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈ and X₉, these substituents may be the same or different and areselected from hydrogen or lower alkyl, i.e. a C₁₋₅ alkyl such as methyl,ethyl or n-propyl. In addition, paired substituents X₁ and X₄ or X₅, X₂or X₃ and X₆ or X₇, X₄ or X₅ and X₈ or X₉, when taken together with thethree adjacent carbon atoms of the central part of the compound, whichcorrespond to positions 8, 14, 13 or 14, 13, 17 or 13, 17, 20respectively, can be the same or different and form a saturated orunsaturated, substituted or unsubstituted, carbocyclic 3, 4, 5, 6 or 7membered ring.

Preferred compounds of the present invention may be represented by oneof the following formulae:

In the above formulae Ib, Ic, Id, Ie, If, Ig and Ih, the definitions ofY₁, Y₂, R₆, R₈, R, Z, X₁, X₂, X₃, X₄, X₅, X₆, X₇ and X₈ are aspreviously set forth herein. The substituent Q represents a saturated orunsaturated, substituted or unsubstituted, hydrocarbon chain comprisedof 0, 1, 2, 3 or 4 carbon atoms, but is preferably the group —(CH₂)_(k)—where k is an integer equal to 2 or 3.

Methods for making compounds of formulae Ia-Ih are known. Specifically,reference is made to International Application Number PCT/EP94/02294filed 7 Jul. 1994 and published 19 Jan. 1995 under InternationalPublication Number WO95/01960.

1-48. (canceled)
 49. A method of treating acne comprising administeringto a patient with acne an effective amount of20(S)-1α,25-dihydroxy-2-methylene-26,27-dihomo-19-norvitamin D₃ havingthe structure


50. The method of claim 49 wherein the compound is administered orally.51. The method of claim 49 wherein the compound is administeredparenterally.
 52. The method of claim 49 wherein the compound isadministered transdermally.
 53. The method of claim 49 wherein thecompound is administered topically.
 54. The method of claim 49 whereinsaid effective amount comprises about 0.01 μg/day to about 100 μg/day of20(S)-1α,25-dihydroxy-2-methylene-26,27-dihomo-19-norvitamin D₃.
 55. Themethod of claim 49 wherein said effective amount comprises about 0.1μg/day to about 50 μg/day of20(S)-1α,25-dihydroxy-2-methylene-26,27-dihomo-19-norvitamin D₃.
 56. Amethod of treating acne comprising administering to a patient with acnean effective amount of20(S)-26,27-dimethylene-25-methoxy-2-methylene-19-norvitamin D₃ havingthe structure


57. The method of claim 56 wherein the compound is administered orally.58. The method of claim 56 wherein the compound is administeredparenterally.
 59. The method of claim 56 wherein the compound isadministered transdermally.
 60. The method of claim 56 wherein thecompound is administered topically.
 61. The method of claim 56 whereinsaid effective amount comprises about 0.01 μg/day to about 100 μg/day of20(S)-26,27-dimethylene-25-methoxy-2-methylene-19-norvitamin D₃.
 62. Themethod of claim 56 wherein said effective amount comprises about 0.1μg/day to about 50 μg/day of20(S)-26,27-dimethylene-25-methoxy-2-methylene-19-norvitamin D₃.
 63. Amethod of treating acne comprising administering to a patient with acnean effective amount of20(S)-1α,25-dihydroxy-26,27-dimethylene-2-methylene-19-norvitamin D₃having the structure


64. The method of claim 63 wherein the compound is administered orally.65. The method of claim 63 wherein the compound is administeredparenterally.
 66. The method of claim 63 wherein the compound isadministered transdermally.
 67. The method of claim 63 wherein thecompound is administered topically.
 68. The method of claim 63 whereinsaid effective amount comprises about 0.01 μg/day to about 100 μg/day of20(S)-1α,25-dihydroxy-26,27-dimethylene-2-methylene-19-norvitamin D₃.69. The method of claim 63 wherein said effective amount comprises about0.1 μg/day to about 50 μg/day of20(S)-1α,25-dihydroxy-26,27-dimethylene-2-methylene-19-norvitamin D₃.70. A method of treating acne comprising administering to a patient withacne an effective amount of20(S)-26,27-dimethylene-1α-hydroxy-2-methylene-24-dehydro-19-norvitaminD₃ having the structure


71. The method of claim 70 wherein the compound is administered orally.72. The method of claim 70 wherein the compound is administeredparenterally.
 73. The method of claim 70 wherein the compound isadministered transdermally.
 74. The method of claim 70 wherein thecompound is administered topically.
 75. The method of claim 70 whereinsaid effective amount comprises about 0.01 μg/day to about 100 μg/day of20(S)-26,27-dimethylene-1α-hydroxy-2-methylene-24-dehydro-19-norvitaminD₃.
 76. The method of claim 70 wherein said effective amount comprisesabout 0.1 μg/day to about 50 μg/day of20(S)-26,27-dimethyl-1α-hydroxy-2-methylene-24-dehydro-19-norvitamin D₃.